Pancreatic progenitor cells represent an important resource for cell replacement therapy in diabetes, and may also represent the cell-of-origin for pancreatic cancer. Among the many transcription factors regulating pancreatic development and differentiation, expression of the basic helix-loop-helix transcription factor, ptf1a-p48, represents the defining feature of pancreatic progenitor cells. Several laboratories, including our own, have identified distinct early and late effects of ptf1a-p48 during pancreatic development. Early in development, ptf1a-p48 is widely expressed in the emerging pancreatic epithelium, where it is required for specification of pancreatic identity, as well as for early growth and morphogenesis. Later in development, ptf1a-p48 becomes restricted to the exocrine compartment, where it induces cell cycle exit and acinar cell differentiation. However, the mechanisms by which ptf1a-p48 exerts its different developmental stage-specific effects remain unknown. We now propose to comprehensively characterize both early and late ptf1a-p48 functions, taking advantage of our unique ability to study pancreas development in both mouse and zebrafish. This work will be based on the following central hypotheses: First, that the early and late effects of ptf1a-p48 on pancreatic development are mediated through entirely different sets of ptf1a-p48 target genes; second, that different structural domains of the ptf1a-p48 molecule may be required for induction of these early and late effects; and third, that ptf1a-p48 target genes may include both coding and non-coding elements, including functionally important microRNA's. To test these hypotheses, the following Specific Aims will be pursued: 1) To identify discrete domains of the ptf1a-p48 molecule responsible for mediating its developmental stage-specific effects on pancreatic development, using newly developed in vivo assays; 2) To identify novel ptf1a-p48 target genes through genome-wide ChIP-on-chip analysis, and determine gain-of-function and loss-of-function phenotypes associated with altered expression of these genes; and 3) To identify and functionally characterize novel ptf1a-p48-regulated microRNA's in developing mouse and zebrafish pancreas. Together, these studies will determine the mechanisms by which ptf1a-p48 exerts its multiple influences in developing mouse and zebrafish pancreas. In so doing, it is likely that we will also identify important new regulators of pancreatic specification, morphogenesis and differentiation. By clarifying the role of this important transcription factor in regulating the pancreatic progenitor pool, these studies will contribute to the eventual therapeutic manipulation of these cells in the context of pancreatic cancer and diabetes. Public Health Relevance: Pancreatic progenitor cells represent an important resource for cell replacement therapy in diabetes, and may also represent the cell-of-origin for pancreatic cancer. Studies in this proposal focus on how Ptf1a-p48, a pancreatic transcription factor, regulates the initial specification, growth and differentiation of pancreatic progenitor cells. We have recently completed two genome-wide screens for genes that may act downstream of Ptf1a-p48. In so doing, we have identified a number of additional coding and non-coding genes that may play important roles in regulating the pancreatic progenitor pool. Using both mouse and zebrafish model systems, we now plan to functionally characterize these novel Ptf1a-p48 target genes. By better clarifying how Ptf1a-p48 regulates progenitor cell growth and differentiation, these studies will contribute to the eventual therapeutic manipulation of pancreatic progenitors in the context of pancreatic cancer and diabetes.